Engineering a Fluorescent Reporter Cell Line to Monitor Ccl19 Expression

Authors:

Sandhya Konar, Karin Gustafsson, David Scadden

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

The thymus plays a critical role in T-cell development by CRISPR effector constructs, which we then collected and used supporting the recruitment and differentiation of lymphoid to infect both HEK 293T and NIH 3T3 cells. Before applying progenitors from bone marrow. This process is orchestrated by selection, we set up an antibiotic kill curve to determine the thymicstromalcells,whichproducesignalingmoleculesthatguide optimal drug concentrations needed to eliminate uninfected cells. immune cell trafficking and development. Recent work from our Following this, we applied antibiotic selection to enrich cells that labidentifiedCcl19, achemokinesecretedbythymicstromalcells, had successfully integrated the constructs using two antibiotics: as a key factor in recruiting these progenitors. While the functiongromycin and blasticidin. We are currently monitoring the of Ccl19 is increasingly well understood, the upstream regulatorssurvival and expansion of these cells to confirm stable expression thatcontrolitsexpressionremainlargelyunknown. Understanding of CRISPRa/i components. Once validated, the effector cell lines how Ccl19 is regulated could provide new insights into thymic will be transduced with single-guide RNAs (sgRNAs), which function, immune reconstitution, and age-related immune decline. will target the Ccl19 promoter to modulate gene expression. To address this, we aimed to develop a fluorescent reporter OverexpressionwillthenbevalidatedbyquantifyingCcl19mRNA cell line to monitor Ccl19 expression and use it as a platform and protein levels. Ultimately, this system will support high- for genome-wide CRISPR screens. To begin, we transfected throughput CRISPR-based screens to uncover novel upstream HEK 293T and NIH 3T3 cells with lentiviral plasmids encoding regulators of Ccl19 and improve our understanding of thymic CRISPRa and CRISPRi effectors (dCas9-VP64, MPH, and dCas9- stromal regulation in immune development. KRAB). This enabled us to generate viral supernatants containing

Abstract:

The thymus plays a critical role in T-cell development by CRISPR effector constructs, which we then collected and used supporting the recruitment and differentiation of lymphoid to infect both HEK 293T and NIH 3T3 cells. Before applying progenitors from bone marrow. This process is orchestrated by selection, we set up an antibiotic kill curve to determine the thymicstromalcells,whichproducesignalingmoleculesthatguide optimal drug concentrations needed to eliminate uninfected cells. immune cell trafficking and development. Recent work from our Following this, we applied antibiotic selection to enrich cells that labidentifiedCcl19, achemokinesecretedbythymicstromalcells, had successfully integrated the constructs using two antibiotics: as a key factor in recruiting these progenitors. While the functiongromycin and blasticidin. We are currently monitoring the of Ccl19 is increasingly well understood, the upstream regulatorssurvival and expansion of these cells to confirm stable expression thatcontrolitsexpressionremainlargelyunknown. Understanding of CRISPRa/i components. Once validated, the effector cell lines how Ccl19 is regulated could provide new insights into thymic will be transduced with single-guide RNAs (sgRNAs), which function, immune reconstitution, and age-related immune decline. will target the Ccl19 promoter to modulate gene expression. To address this, we aimed to develop a fluorescent reporter OverexpressionwillthenbevalidatedbyquantifyingCcl19mRNA cell line to monitor Ccl19 expression and use it as a platform and protein levels. Ultimately, this system will support high- for genome-wide CRISPR screens. To begin, we transfected throughput CRISPR-based screens to uncover novel upstream HEK 293T and NIH 3T3 cells with lentiviral plasmids encoding regulators of Ccl19 and improve our understanding of thymic CRISPRa and CRISPRi effectors (dCas9-VP64, MPH, and dCas9- stromal regulation in immune development. KRAB). This enabled us to generate viral supernatants containing

Source:

Harvard / Juliette Gaytan, Viral S. Shah, Jayaraj Rajagopal / 2025

Topics:

cell, ccl19, expression, immune, line, development, crispr, effector, antibiotic, fluorescent, reporter, monitor

Co-authors:

@sandhyakonar199 , @karingustafsson200 , @davidscadden201